Intra-Species Diversity of Leishmania major and L. tropica from Clinical Isolates of Cutaneous Leishmaniasis in Southwest Iran Inferred by ITS1-rDNA.

Background
Cutaneous leishmaniasis (CL) as a public health concern is increasingly circulating by causative agents of Leishmania tropica and L. major in Iran. As regard to recent treatment failure and controlling problems, the accurate elucidation of heterogeneity traits and taxonomic status of Leishmania spp. should be broadly addressed by policymakers. This study was designed to determine the genetic variability and molecular characterization of L. major and L. tropica from Iranian CL patients.


Methods
One hundred positive Giemsa-stained slides were taken from clinical isolates of CL at Pol-e-Dokhtar County, Southwest Iran, from May 2014 to Sep 2016. DNAs were directly extracted and amplified by targeting ribosomal internal transcribed spacer (ITS) gene following microscopic observation. To identify Leishmania spp. amplicons were digested by restriction enzyme HaeIII subsequent PCR-RFLP technique. To reconfirm, the isolates were directly sequenced to conduct diversity indices and phylogenetic analysis.


Results
Based upon the RFLP patterns, 84 and 16 isolates were explicitly identified to L. tropica and L. major respectively. No co-infection was found in clinical isolates. The high genetic diversity of L. tropica (Haplotype diversity 0.9) was characterized compared to L. major isolates (Hd 0.476). The intra-species diversity for L. tropica and L. major isolates corresponded to 3%-3.9% and 0%-0.4%, respectively.


Conclusion
Findings indicate the L. tropica isolates with remarkable heterogeneity than L. major are predominantly circulating at Pol-e-Dokhtar County. Occurrence of high genetic variability of L. tropica may be noticed in probable treatment failure and/or emerging of new haplotypes; however, more studies are warranted from various geographic regions of Southwest Iran, using large sample size.


Introduction
Cutaneous leishmaniasis (CL) is a group of neglected vector-borne disease which presents by various clinical appearances and heterogeneity traits (1,2). CL is dispersed in 20 out of 31 Irani-an provinces with overall prevalence of 1.8% to 37.9% and estimated annual incidence of 26630 cases (2)(3)(4). In order of clinical-epidemiology importance, two well-known etioparasitological agents, Leishmania major, and L. tropica are principal causative agents of CL in Iran (3,5). The genetic diversity of the genus Leishmania is discussed as one of the most controversial issues among the endemic areas. The genetic heterogeneity of Leishmania parasite may lead to emergence of diverse phenotypic aspects, emergent sub-species/strains, and formation of novel haplotypes, and subsequently rapid generation of drug-resistance alleles (6)(7)(8)(9). Currently, L. major and L. tropica are unambiguously circulating in Lorestan province, where it is addressed as a neglected focus of leishmaniasis in Southwest Iran (10). To evaluate genetic diversity examination of Leishmania spp. several nuclear and mitochondrial DNA genes have tested, including Cytochrome b (kDNA maxicircle), microsatellites, gp63, 18S-rRNA, mini-exon, HSP-70 and ribosomal internal transcribed spacer regions (ITS-rDNA) (3,(11)(12)(13)(14)(15)(16)(17). The ITS-rDNA region can utilize to un-derstand evolutionary hypotheses of Leishmania as a result of low intracellular polymorphism and to be conserved regions (18). As regard to recent drug resistance and controlling problems, the precise elucidation of single nucleotide polymorphism (SNP) and taxonomic status of Leishmania spp. by more sensitive strategies could be generally noticed by regional policymakers. The aim of this study was to determine molecular characterization and genetic variability of L. major and L. tropica from Pol-e-Dokhtar County, Southwest Iran.

Study area
Pol-e-Dokhtar County is located in the South of the Lorestan Province ( Fig.1). This county occupied 3615 square km, and according to the Statistical Center of Iran (SCI), the population size is 75,327. Pol-e-Dokhtar city has an altitude of 673 meters above sea level. The annual average temperature of the city is 11.9ºC.

Sampling and microscopic examination
The lesions of suspected patients to CL were sampled from Pol-e-Dokhtar Health Center, from May 2014 to Sep 2016. Overall, 100 microscopically confirmed slides were collected. The samples were smeared on microscopic slides, dried, and stained by Giemsa. Leishmania infections were identified under light microscope with high magnification (×1000). Demographic and clinical data of each patient including age, sex, locality, the number of lesions, and location of lesion were recorded using a questionnaire.

Extraction of Total Genomic DNA, PCR amplification and restriction fragment length polymorphism for ITS1-rDNA
DNA of Leishmania was directly extracted from positive slides based upon the Phenolchloroform protocol (6). All Giemsa-stained slides were washed with ethanol and covered with 250μL lysis buffer (50mM NaCl, 50 mMTris, 10mM EDTA, pH 7.4, 1% v/v Triton x-100 and 100 μg of proteinase k per ml). To accomplish, cell lysis samples were then incubated at 56ºC for 3 h or overnight. The obtained DNA was resuspended in 30 μL double-distilled water and stored at -20 ºC until molecular doings. The single round PCR was performed to identify the Leishmania infection by targeting ribosomal internal transcribed spacer 1 (ITS1) using previously designed specific primers, LITSR (5`-CTGGATCATTTTCCGATG-3`) as Forward primer and L5.8S (5`TGATACCACTTATCGCACTT-3`) as reverse primer (19). The following program was used to carry out PCR: initial denaturing cycle at 94°C for 5 min, followed by 35 cycles of 94ºC for 30 sec, 47 ºC for 30 sec, 72 ºC for 1 min and finally 1 cycle of 72ºC for 5 min. PCR products were run on 1.5%gel agarose stained with ethidium bromide and observed under UV light. Restriction fragment length polymorphism (RFLP) was performed on PCR products to determine the parasite species. Endonuclease reaction of ITS1-rDNA gene was done in a volume of 30 L containing 2 L of BsuRI (HaeIII) with cut site GG↓CC, 10 L of PCR products, 2 L of 10x buffer, and 16 L of distilled water for 15 min at 37°C. Finally, the digested fragments were revealed using 3% gel agarose and UV light.

Sequencing and phylogenetic analyses
To re-confirm the RFLP findings, amplicons were randomly purified and sequenced by targeting ITS1-rDNA gene using ABI 3130X automatic sequencer at the Bioneer Company, South Korea. Contigs (overlapped sequences) were aligned (http://multalin.toulouse.inra.fr/multalin) and edited at consensus positions using Sequencher Tmv.4.1.4 Software (Gene Codes Corporation). The percent identity and divergence (pairwise distances) among sequenced isolates were drawn using the MegAlign program from Laser Gene Bio Computing Software Package (DNASTAR, Madison, WI). To demonstrate the taxonomic status of identified isolates, the phylogenetic tree was constructed by MEGA 5.05 software based on Maximum Likelihood algorithm and kimura two-parameter model. The accuracy of phylogenetic tree was evaluated by 1000 bootstrap re-sampling. The Trypanosoma brucei (Accession number: JN673390) and Leishmania mexicana (Accession number: AF466383) were considered as out-group branches. The diversity indices (haplotypes diversity [Hd] and nucleotide diversity [π]) was calculated by DnaSP software version 5.10 (20).

Results
The demographic and clinical data of CL cases based on sex, age group, and location of lesion are given in Table 1. The vast majority infection was identified in 20-29 yr olds (25%) than other groups and in males (70%). The most lesions located at hand (53%) and foot (26%) ( Table 1). The ITS1-rDNA gene (target 361bp) was successfully amplified by single round PCR for all Leishmania isolates.
The in-silico prediction of BsuR1 restriction enzyme for ITS1-rDNA gene of L .major and L. tropica is shown in Fig. 2A. Based upon the RFLP digested patterns, 84 and 16 isolates were identified to L. tropica (fragments 57bp, 56bp and 200bp) and L. major (fragments 155bp and 206bp) isolates, respectively (Fig. 2 B-C). No coinfection was found in identified isolates. The high genetic diversity of L. tropica (Hd 0.9) was characterized compared to L. major (Hd 0.476).  L. tropica sequences (0.02176) had more nucleotide diversity than L. major isolates (0.00183) ( Table 2). The multiple sequence alignment of ITS1-rDNA gene for L .major and L. tropica is shown in Fig. 3. The intra-species diversity and identity for L. tropica isolates were 3-3.9% and 96-97.3% while, for L. major isolates corresponded to 0%-0.4% and 99.6%, respectively (Fig. 4). The topol-ogy of constructed phylogenetic tree disclosed that the L. major (Accession nos: KY612600-KY612607) and L. tropica (Accession nos: KY612608-KY612611) were grouped with bootstrap value of higher than 60% in their specific complex (Fig. 5). The Trypanosoma brucei and Leishmania mexicana were considered as out-group branches.

Discussion
In this investigation, different levels of genetic diversity of L. tropica and L. major isolates were identified in clinical isolates of CL patients from Southwest Iran, inferred by ITS1-rDNA nucleus gene. L. tropica (n=86%) was firmly determined by RFLP and phylogenetic analyses as the principal species responsible for CL in the Pol-e-Dokhtar County, where here was not an eligible information concerning taxonomic status and heterogeneity traits of parasite causing CL (10).
Pol-e-Dokhtar County is known as a neglected hyperendemic focus of leishmanisis, which neighbored with Khuzestan and Ilam provinces. Current finding shows that the most CL infections were occurred in males with age group 20-29 yr old. This may be males frequently work in the field places and females cover their body and apply Hijab because of religion (3). In this study, nuclear ribosomal internal transcribed spacer region as a universal DNA barcode marker was used to identify probable haplotypes and evolutionary relationship of L. tropica and L. major because of its conserve features and be multi-copy (18).
In the current findings, the L. tropica isolates demonstrated a greater genetic diversity (Hd: 0.9 and nucleotide diversity; 0.02176) than L. major (hd: 0.476 and nucleotide diversity: 0.00183). This may be described by the genomic characters of ITS-rDNA. GC content of L. tropica is generally lower than L. major (59.7%), therefore the stability of triple hydrogen bonds of the GC pair and stacking interaction is faced with a slippage (21,22). Furthermore, this heterogeneity may elucidate by several facts; First: presence of turnover mechanisms; unequal crossing over/transposition and slippage in the sequence length of parasite (23). Second: lack of any bottleneck effects.
One of the limitations of current investigation is that the study area was highly confined and the isolates are relatively small to be inferred for more extensive larger-scale genetic diversities, However; we cannot explicitly estimate the heterogeneity levels of L. tropica and L. major in the region. On the one hand, the employing evolutionary mitogenome markers (e.g. Cytochrome b or Cyt c oxidase subunit I) can identify the new haplotypes than nuclear genes (e.g., ITS1-rDNA).
Up to now, the genetic diversity of Leishmania spp. have been reported by several regional researchers by targeting nucleus (ITS-rDNA) and/or mitochondrial (kDNA and Cyt b) markers from various endemic foci of Iran. The sequence alignment of kDNA gene demonstrated a high genetic diversity of L. major from different rural and urban areas of Fars Province (Southern Iran) (24). However, by targeting ITS-rDNA gene a high genetic polymorphism of L. major (7.3%) than L. tropica isolates (3.6%) had shown in various locations of Iran (25) (29).The genetic diversity in L. major strains was determined belong to different endemic areas of Iran using kDNA, there was considerable diversity between strains of different regions and even between isolates that belong to the same area (30). This study revealed that both of main causative agents of CL are present in Pol-e-Dokhtar County. In accordance with previous studies, L. tropicawas found predominant species in the study area (10,31). L. tropicaand L. major were the main causative agents of CL in Pol-e-Dokhtar County with the occurrence of 82.2 and 35.3% respectively (10,31). In this study, the most affected part of the body was belonged to hand, foot, and face, respectively. This finding is in accordance with the study conducted in the same area (31).

Conclusion
The L. tropica isolates with remarkable heterogeneity than L. major are predominantly circulating at Pol-e-Dokhtar County. Occurrence of high genetic variability of L. tropica may be noticed in probable treatment failure and/or emerging of new haplotypes; however, more studies are war-ranted from various geographic regions of Southwest Iran, using large sample size.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.